Small interfering RNA (siRNA) molecules are pieces of RNA that block the activity of genes through a natural process called RNA interference (RNAi). RNAi has emerged as a powerful tool used in thousands of labs worldwide to understand gene function. By blocking or modulating a genes function, RNAi can tell us about the role of any gene in maintaining health or causing disease, an invaluable step in identifying potential drug targets. In tests called genome-wide RNAi screens, scientists use automation to introduce siRNAs into human cells to block the activity of each gene, one at a time. This process can produce a complete list of all genes involved in a particular biological function or disease process. Scientists also can use these techniques to understand what roles genes play in drug effectiveness. RNAis potential usefulness has been limited by the lack of expertise to perform genome-wide RNAi screens, the lack of methodologies that can properly interpret these experiments and the absence of comprehensive RNAi data in public databases for researchers to reference. To address these problems, NCATS operates a state-of-the-art RNAi screening facility known as the Trans-NIH RNAi Facility (TNRF), and NCATS staff assists NIH intramural investigators with all stages of project planning and execution. The initiative provides public access to RNAi data generated from these experiments through the National Library of Medicines PubChem database. In addition, siRNA sequence information is available from private-sector biotechnology partners. For instance, researchers can access Life Technologies Silencer Select siRNA library, which includes 65,000 siRNA sequences that target more than 20,000 human genes. The RNAi high-throughput screening facility, administered by NCATS Division of Pre-Clinical Innovation staff, offers a robotic platform with integrated, automated devices for conducting all aspects of screening assays (tests), including manipulating chemicals and cells, reading the results and imaging the cells. Offline (non-robotic) devices can perform smaller-scale work from assay optimization through medium-scale screening. Investigators have the option of using several different siRNA libraries and other small molecules involved in RNAi. For data analysis, the facility offers powerful computational tools. In addition to enabling collaborations on specific projects, TNRF staff work to develop methods that advance the science of RNAi screening and informatics and pursue new technologies for exploring gene function. Currently, NCATS scientists are working on developing a platform that offers complementary high-throughput genomic technologies to advance the field of functional genomics. They are performing multiple screens in order to optimize this platform. The goal is to speed scientific discoveries in all disease areas. Project areas include cancer (drug enhancer/resistance screens, development of 3D metastasis screens, molecular targets in cancer, and cancer-related pathways), infectious diseases (viral infection and replication such as Zika virus, HIV, Ebola virus, and Hepatitis C virus), fundamental cell biology (DNA replication and reprogramming/differentiation), and other disease-related phenotypes (Parkinsons disease, diabetes, and fragile X syndrome).